Below is a summary of our recent findings for each major research area. 1) Develop novel therapeutics and diagnostics against cancer and human immunodeficiency virus type 1 (HIV-1). We have hypothesized that the smallest independently folded antibody fragments (domains) could exhibit exceptionally potent and broadly cross-reactive neutralizing activity by targeting hidden conserved epitopes on the HIV-1 envelope glycoprotein (Env) that are not accessible by larger antibodies. To test this hypothesis we constructed a novel type of large (size 2.5 x 1010) highly diversified library of human antibody variable domains (domain antibodies) and used it for selection of binders to conserved Env structures by panning sequentially against Envs from different isolates. The highest affinity binder, m36, neutralized all tested HIV-1 isolates from clades A, B, C, and D with an activity on average higher than that of C34, a peptide similar to the fusion inhibitor T20 which is in clinical use, and that of m9 which exhibits a neutralizing activity superior to known potent cross-reactive antibodies. M36 is the first representative of a novel class of potent, broadly cross-reactive HIV-1 inhibitors based on human domain antibodies. It has potential for prevention and therapy, and as an agent for exploration of the closely guarded conserved Env structures with implications for design of small molecule inhibitors, and elucidation of mechanisms of virus entry and evasion of immune responses. We have also engineered CH2 domain antibodies to confer antigen binding properties in addition to full or partial effector functions (nanoantibodies). Work is in progress to further develop this new concept for the development of novel therapeutics and diagnostics against cancer and HIV-1. 2) Develop an effective acquired immunodeficiency syndrome (AIDS) vaccine using knowledge obtained from the human antibodyome. We have proposed to sequence large portions of the human antibody repertoire which we called the Antibodyome Project funded jointly by NIAID, the Bill and Melinda Gates Foundation, and Center for HIV-AIDS Vaccine Immunology (CHAVI, Duke University, Barton Haynes, Director). This proposal was initially based on our observation that HIV-1-specific cross-reactive neutralizing antibodies (crnAbs) are highly divergent from the closest germline in contrast to crnAbs against viruses causing acute infections such as the henipaviruses Nipah (NiV) and Hendra (HeV) and the severe acute respiratory syndrome (SARS) CoV. We found that the antibody somatic mutational diversification (ASMD of all known HIV-1-specific crnhmAbs was significantly higher ( 3-30-fold) than that for crnhmAbs against the SARS CoV and henipaviruses. The ASMD rate of two newly selected antibodies from an acutely HIV-1-infected patient was about two mutations per month. The ASMD rate for a panel of SARS CoV-specific antibodies was about one mutation per month. Using a linear interpolation based on these data it was estimated that elicitation of known HIV-1-specific crnhmAbs could require 3 years. Thus elicitation of HIV-1-specific crnhmAbs may require extensive ASMD in contrast to the SARS CoV and henipaviruses. We hypothesize that the infrequent occurrence (absence) of crnhmAbs in HIV-1-infected (immunized) individuals is caused in part by a lack or limited availability of B cell receptors that are able to rapidly mature to cross-reactive neutralizing antibodies. Thus, appropriate immunization protocols of long duration should be developed using the knowledge gained from the exploration of the antibody maturation pathways in humans. 3) Develop novel antibody-based therapeutics. We have also continued to characterize our antibodies against components of the insulin-like growth factor (IGF) system which plays an important role in cancer. Currently several monoclonal antibodies targeting the IGF receptor type I (IGF-IR) are being tested in patients with solid tumors. However, penetration of full-length antibodies into solid tumors is slow and inefficient. We have been hypothesizing that targeting the IGF-IR ligands, IGF-I and IGF-II, by antibodies may not require antibody presence in the tumor interstitial space but could shift the complex equilibrium and quasi-steady states to ligand-antibody complexes in the plasma leading to depletion of the ligand in the tumor. We continued to test this hypothesis with a fully human monoclonal antibody, IgG1 m610, which binds with high (nM) affinity to IGF-II and inhibits signal transduction mediated by the IGF-II interaction with the IGF-IR. We matured in vitro m610 and found two antibodies with higher affinity that are being currently characterized. In addition, we identified several other hmAbs against cancer-related proteins including mesothelin and CD22. Mesothelin is a potential new target for cancer immunotherapy because it is present at relatively low levels only in mesothelial cells of pleura, peritoneum and pericardium of healthy people but is significantly elevated in a number of tumors, including mesothelioma, ovarian cancer, pancreatic cancer, and lung cancer. However, all currently available antibodies against mesothelin are either murine or chimeric which limits their use because of increased likelihood of immunogenicity compared to fully human antibodies. We identified and characterized a novel fully human monoclonal antibody, m912, which was isolated from a human Fab library by panning against recombinant mesothelin. This antibody specifically lysed cancer cells engineered to express mesothelin in the presence of peripheral blood mononuclear cells isolated from healthy donors most likely by antibody-dependent cellular cytotoxicity (ADCC). M912 is the first reported fully human monoclonal antibody to mesothelin, which has potential for cancer treatment and diagnosis. We also identified two novel hmAbs against CD22 which have similar properties as those against mesothelin but are specific for CD22 and could be used as potential cancer therapeutics. Responding to the threat of bioterrorism and to the health crisis caused by the SARS coronavirus (SARS CoV), we have continued to identify and characterize neutralizing antibodies to the biodefense-related Hendra (HeV) and Nipah (NiV) viruses, and to the SARS CoV. Previously, we reported on the isolation of henipavirus-neutralizing recombinant hmAbs including one, m102.4, which exhibited exceptionally potent and cross-reactive inhibitory activity against both HeV and NiV. We recently tested this antibody in an animal model based on ferrets and found that ferrets that received m102.4 intravenously ten hours after oral-nasal NiV challenge were protected from disease while all controls died. This study represents the first successful post-exposure passive antibody therapy for NiV where a purified, fully human antibody was employed. These results further confirm our proposition that m102.4 has potential as a therapeutic for treatment of diseases caused by henipaviruses, and could save human lives now. It could be also used for prophylaxis, diagnosis and as a research reagent.